Leveraging the high sensitivity, specificity and quantitative capability of the droplet digital PCR (ddPCR) platform, this project seeks to analytically and clinically validate ddPCR assays for 7 melanoma-associated hot- spot mutations for use as blood-based biomarkers of disease recurrence in patients with resected metastatic melanoma. In the United States, the only commonly used serologic marker to monitor disease recurrence in patients with resected metastatic melanoma is serum lactate dehydrogenase (LDH), which suffers from low sensitivity and specificity. With new, effective therapies for systemic disease being applied in the adjuvant setting, a sensitive and specific biomarker that can identify early disease recurrence could prompt clinicians to change patient management while the patient has a low tumor burden. Recently, highly sensitive and specific technologies, such as ddPCR, have revealed that cancer patients have abnormally high levels of cell-free, tumor-associated DNA (ctDNA) circulating in their plasma that generally correlate positively with metastatic tumor burden. We conducted a pilot study utilizing ddPCR to detect changes in mutant BRAF and NRAS ctDNA levels in patients with unresectable stage IV melanoma who were undergoing treatment with BRAF- targeted therapies or immune checkpoint blockade. Importantly for this application, we found that at the time of treatment initiation, ctDNA was a much more sensitive marker of low disease burden than LDH. ctDNA levels were elevated in 71% of patients with RECIST scores < 5cm at the start of systemic treatment compared to LDH, which was only elevated in 8% of patients. While these encouraging results were obtained with assays that detect 1 of the 5 of the most common melanoma-associated BRAF and NRAS mutations, collectively these 5 mutations are present in only 55% - 65% of patients. Recently, 2 functional, hotspot mutations in the promoter region of the telomerase reverse-transcriptase gene (TERT) were identified in many cancers including melanoma. Our preliminary studies have also found these TERT mutations frequently in melanoma and when evaluated with BRAF and NRAS mutations, 86% of patients have 1 or more of the 7 BRAF, NRAS or TERT mutations in their melanomas. In this application, we will analytically validate ddPCR assays for the 7 common mutations in BRAF, NRAS and TERT, and test the hypothesis that a rise in ctDNA levels, as measured by 1 of these 7 ddPCR assays will predict radiographic recurrence in resected metastatic patients prior to routine imaging scans. Development of plasma biomarkers that can accurately detect disease progression with high sensitivity can augment or replace the routine radiographic scans obtained every 3 months with scans ordered in response to a rise in the plasma ctDNA level. Also it could lay the groundwork for a clinical trial comparing the utility of adjuvant therapy for all resected patients versus selecting patients for treatment when evidence of micrometastatic disease emerges based on a rise in ctDNA levels.